Experimental autoimmune encephalomyelitis (EAE) is a CD4+ Th1/17 cell-mediated inflammatory CNS demyelinating disease that serves as a model for human multiple sclerosis (MS). Disease progression (i.e., relapses) in the SJL/J mouse is characterized by de novo activation of CD4+ T cell responses against non- crossreactive endogenous epitopes on the same or different myelin proteins (intramolecular or intermolecular epitope spreading). Effective specific therapy of established disease requires tolerogenic targeting of T cells specific for spread myelin epitopes. Tolerance can be effectively induced to ameliorate pre-established disease by spread myelin peptide-pulsed, ethylene carbodiimide (ECDI)-fixed splenic APCs (Ag-SP) indicating that responses to spread epitopes play the major pathologic role in mediating disease relapses. Ag-SP- induced tolerance is significantly more efficient and safer than tolerance induced by free peptide or peptide multimers, works primarily by indirect (cross-tolerance) representation of peptide coupled to apoptotic Ag-SP by splenic marginal zone (MZ) APCs which induce unresponsiveness via two separate but complimentary mechanisms: PD-L1/IL-10-dependent cell intrinsic anergy and activation of induced Tregs (iTregs). The experiments proposed in this renewal will test the hypothesis that the uptake of apoptotic Ag-SP by scavenger receptors on splenic MZ macrophages (MZM) induces a 'tolerogenic signature' in the APCs leading to antigen representation (cross-tolerance) and induction of both clonal anergy and activation of antigen-specific CD4+CD25+ iTregs which together regulate both nave and activated encephalitogenic Th1/17 cells. Aim 1 of the proposal will build on our productive studies from the previous funding period by further defining the phenotype and functional capacity of MZ APCs involved in interaction with and tolerogenic cross-presentation of Ag-SP resulting in the antigen-specific regulation of encephalitogenic Th1 and Th17 responses. The role of scavenger receptors in Ag-SP uptake and the innate response of the tolerogenic APCs will be elucidated. Aim 2 will test the hypothesis that maintenance of Ag-SP tolerance is primarily due to the activity of antigen-specific iTregs. The ability of Ag-SP t activate iTregs both in vivo and in vitro will be assessed, as will their role in maintenance of Ag SP tolerance and their potential ability to differentially regulate effector functions of nave and activated Th1 vs. Th17 cells. Aim 3 will test the hypothesis that uptake of Ag-SP by tolerogenic splenic MZ APCs induces a cell intrinsic anergy program in nave and activated Th1/17 cells due to engagement of regulatory costimulatory molecules (CTLA-4/PD-L1) and/or cytokines (IL-10/TGF-) which results in inhibition of normal Th1/17 signaling pathways resulting in inhibition of their differentiation/effector function. Collectively, these studies should enhance the understanding of the cellular/molecular mechanisms underlying a highly efficient antigen-specific tolerance therapy which is currently being tested in a MRI-controlled Phase I/IIa clinical trial in new-onset relapsing-remitting MS patients.